The present invention relates to binding proteins, methods of making the proteins, and uses thereof. More particularly, the present invention relates to synthetic binding proteins which comprise immunoglobulin heavy and light chain variable domain binding regions. Polypeptides are provided which form multimers, eg dimers, which may have more than one binding specificity.
Because of the length of the specification, it is appropriate to include here a list of contents to help the reader find passages of interest. In addition to claims, figures and an abstract, the specification includes the following, in order:
Statement of the field of the invention;
Contents;
Brief discussion of prior art and introduction to the invention;
(Background)
Discussion of the invention;
Summary of the Invention
Brief description of the figures;
Discussion of bivalent and bispecific antibodies, preparation and uses;
Preparation of xe2x80x9cdiabodiesxe2x80x9d, discussion of structure and various utilities;
Discussion of construction of repertoires of diabodies and their display on bacteriophage;
Listing of the experimental examples;
Experimental examples and discussion;
All documents mentioned in this text are incorporated herein by reference.
Natural antibodies are multivalent, for example Ig G antibodies have two binding sites and IgM antibodies have five binding sites for antigen. The multivalency means that the antibodies can take advantage of multiple interactions in binding to solid phase antigen, and therefore increasing the avidity of binding to the antigen. It is possible to make recombinant bivalent IgG and pentameric decavalent IgM antibodies by expression in mammalian cells. To date, of the various possibilities for multivalency bivalent antibodies have been of greatest interest.
Of further interest are antibodies which are able to bind to two or more different epitopes, those which have multispecificity. Bispecific antibodies have many proven and expected utilities, discussed infra.
Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds. The light chains exist in two distinct forms called kappa (K) and lambda (xcex). Each chain has a constant region (C) and a variable region (V). Each chain is organised into a series of domains. The light chains have two domains, corresponding to the C region and to the V region. The heavy chains have four domains, one corresponding to the V region and three domains (1, 2 and 3) in the C region.
The antibody has two arms (each arm being a Fab region), each of which has a VL and a VH region associated with each other. It is this pair of V regions that differs from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognising the antigen and providing an antigen binding site. In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of the variable regions, and they perform the critical antigen binding function.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; and (vi) F(abxe2x80x2)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region.
The fragments largely represent portions of complete antibodies. However, the term xe2x80x9cfragmentxe2x80x9d is also applied to synthetic molecules which comprise antibody heavy and light chain variable domains, or binding portions of these domains, associated so as to have specific antigen binding capability. A good example of an antibody fragment which is of this type is the xe2x80x9csingle chain Fvxe2x80x9d (scFv) fragment which consists of an antibody heavy chain variable domain linked to an antibody light chain variable domain by a peptide linker which allows the two domains to associate to form a functional antigen binding site (see, for example U.S. Pat. No. 4,946,778, Ladner et al., (Genex); WO 88/09344, Creative Biomolecules, Inc/Huston et al.). WO 92/01047, Cambridge Antibody Technology et al./McCafferty et al., describes the display of scFv fragments on the surface of soluble recombinant genetic display packages, such as bacteriophage.
Some experimental work has been described wherein the length of the peptide linker of scFv molecules was varied. The range of linker lengths has in general been from 12 to 18 amino acids, with 15 amino acids being the most usual, see e.g. M C Whitlow et al., Protein Engineering 6: 989-995, 1993. In all cases the linker was long enough for the two domains, one VL, the other VH, to associate to form an antigen binding site, this being a characterising feature of scFv""s. Condra et al., for example (The Journal of Biological Chemistry, 256:2292-2295, 1990) varied the length of the linker of scFv fragments able to block human rhinovirus attachment to cellular receptors. What the authors describe in this paper as a VL domain includes amino acids from the C domain (as can be seen from reviewing Kabat et al., Sequences of proteins of Immunological Interest, US Government Printing Office), and these therefore molecules have xe2x80x9clongxe2x80x9d linkers between the variable domains, allowing the VL and VH domains in each molecule to associate to form an antigen binding site.
The present invention contributes further to the field of antibody fragments by providing polypeptides which are able to associate to form multivalent or multispecific multimers, particularly dimers. As discussed in greater detail below, in one aspect the present invention provides a polypeptide which comprises a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the domains being linked but incapable of associating with each other to form an antigen binding site. Such polypeptides are able to associate to form multimers, such as dimers, the heavy chain binding region of one polypeptide associating with the light chain binding region of another polypeptide to form an antigen binding site. In the case of dimerisation two antigen binding sites are formed.
The term xe2x80x9cdiabodyxe2x80x9d has been coined to describe dimers according to the present invention. This term has already gained some recognition in the art. When used in the present application, the term is not intended to be construed in general discussion as applying only to dimers, except where context demands so. Trimerisation of the polypeptides is feasible, three antigen binding sites then being formed. Also, the term is used in relation to both multivalent and multispecific multimers.
Each domain of the polypeptide may be a complete immunoglobulin heavy or light chain (as the case may be) variable domain, or it may be a functional equivalent or a mutant or derivative of a naturally occuring domain, or a synthetic domain constructed , for example, in vitro using a technique such as one described in WO 93/11236 (Medical Research Council et al./Griffiths et al.). For instance, it is possible to join together domains corresponding to antibody variable domains which are missing at least one amino acid. The important characterising feature is the ability of each domain to associate with a complementary domain to form an antigen binding site. Accordingly, the terms xe2x80x9cimmunoglobulin heavy/light chain variable regionxe2x80x9d should not be construed to exclude variants which do not have a material effect on how the invention works.
A xe2x80x9cderivativexe2x80x9d is a substance which is related to a polypeptide. The derivative may differ by the addition, deletion, substitution or insertion of one or more amino acids, or by the linkage of another molecule. Changes may be made at the nucleotide or protein level. Markers such as enzymes, fluoresceins etc may be linked to the polypeptide.
The first and second domains may be linked without any intervening amino acid residues. Two such molecules will then associate to form an even smaller dimer. Furthermore, it may be that in certain cases the rigidity of molecules of this type means that the binding of antigen to one antigen binding site of the multimer will cause a conformational chance in another binding site in the multimer, a conformational change which may be useful if it enhances or reduces antigen binding, or even changes a catalytic activity of that other binding site.
In other embodiments, the domains of the polypeptide are linked by a peptide linker. The linker may be xe2x80x9cshortxe2x80x9d, consisting of too few amino acids to allow the VL domain of a chain to combine with the VH domain of that chain. This may be less than 10 amino acids, most preferably, 5, 4, 3, 2, or 1. It may be in certain cases that 9, 8, 7 or 6 amino acids are suitable. In some cases it may be xe2x80x9cxe2x88x921xe2x80x9d, ie with the VH and VL domains linked directly together, but with one of them missing an amino acid. In certain cases, the omission of more than one amino acid from one or both of the domains may be feasible.
In still further embodiments, the linker may consist of 10 or more amino acids so that, without a limiting structural feature, the domains of each chain would be able to associate with one another to form an antigen binding site. For instance, the linker may be 15 amino acids or longer. Suitable structural constraints include having cysteine residues within the linker with disulphide bridges joining them to reduce the effective length of the linker, in terms of how far the V domains are apart, by xe2x80x9cloopingxe2x80x9d of the linker. This is discussed further infra. Those skilled in the art will be aware of other possibilities.
A dimer of two of the polypeptides may be bivalent, ie with each antigen binding site able to bind the same epitope. The binding specificity of the VH and the VL domains is determinable when the respective domain is in association with a complementary VL or VH, as the case may be. If the VH and VL domains of the polypeptide are derived from a parent antibody or antibody fragment (however obtained) then two of the polypeptides (identical, or with modification which may differ) will combine to form a bivalent dimer. Of course, the binding regions of polypeptides which will associate to form bivalent dimers may be derived from two different antibodies which bind the same epitope or may be synthetic, or obtained using any of the ways suggested to those skilled in the art.
In other cases, the binding region of an immunoglobulin heavy chain variable region and the binding region of an immunoglobulin light chain variable region may be, when in association with complementary light or heavy chain binding region respectively, able to bind different epitopes from each other. A dimer of two such molecules will be bispecific.
The polypeptide may be fused to additional amino acid residues. Such residues may be a peptide tag, perhaps to facilitate isolation, or they may be a signal sequence for secretion of the polypeptide from a host cell upon synthesis (for which see below). Suitably, secretory leader peptides are used, being amino acids joined to the N-terminal end of a polypeptide to direct movement of the polypeptide out of the cytosol.
The additional amino acids may be a polypeptide domain, such as antibody C domain and/or a surface component of an organism such as a bacteriophage, perhaps a filamentous bacteriophage such as fd or M13. Preferably, the surface component is GIII of bacteriophage fd or the equivalent from another filamentous phase.
A polypeptide according to the invention may in association with another such polypeptide form a dimer, with the binding region of an immunoglobulin heavy chain variable region of each polypeptide being in association with a binding region of an immunoglobulin light chain variable region of the other polypeptide.
In another embodiment, a three chain construct may be formed, the construct have two antigen binding sites. There, a polypeptide according to the present invention is allowed to associate with xe2x80x9cfreexe2x80x9d VH and VL domains to form two antigen binding sites. For instance the three molecules may be (VHA-VLB), VHB and VLA, forming a bispecific construct, or they may be (VH-VL), VH and VL, to form a bivalent construct. This embodiment would be appropriate where the VH and VL domains could associate stably. The constructs would be more flexible than the standard diabody pairing, though still advantageously small in size.
Dimers, whether bivalent or bispecific are specifically encompassed by the present invention. As discussed the two polypeptides in a dimer may be different from one another or they may be the same. Where they are different, the order of the first and second domains (xe2x80x9cVHxe2x80x9d and xe2x80x9cVLxe2x80x9d) may conceivably be the same or different. However, most preferably the N-terminal to C-terminal order of the first and second domains is the same in each polypeptide. A bispecifc dimer might then be represented as N-(VHA-VLB)=C/N-(VHB-VLA)-C or N-(VLA-VHB)-C/N-(VLA-VHA)-C.
Also provided by the present invention are diverse repertoires of the polypeptides. Within such a diverse repertoire, different pairings (or trimerisation etc . . . ) of polypeptides may result in the formation of dimers with different binding specificities, either bivalent or bispecific. Diverse repertoires of dimers are encompassed by the present invention.
The present invention also provides nucleic acid comprising a sequence encoding a polypeptide according to the invention, and diverse repertoires of such nucleic acid. The nucleic acid may comprise nucleic acid of an RNA splice site between nucleic acid encoding the first domain and nucleic acid enoding the second domain.
A self splicing group I intron such as that from Tetrahymena (T. R. Cech Ann. Rev. Biochem. 59:543-568, 1990) may be inserted. Splicing out of the intron occurs at the RNA level leaving behind the internal guiding sequence of the intron, which would encode three amino acids between the two domains of the diabody. The self-splicing may be designed so that the number of amino acids remaining is different, for instance, in Example 24, 5 and 6 amino acid linkers are formed.
Other group I introns or group II self splicing introns may be used. Self splicing introns may be used in combination with reconbination, for example, at the loxP site (see below), in the construction of diabody molecules. For example, a loxP site may be included in a self splicing intron between the two antibody domains of a polypeptide chain. This may, for example, be recombined at the DNA level through a loxP site on another replicon carrying another variable domain gene and the appropriate region of a self splicing intron. Self splicing at the RNA level following transcription will now lead to a diabody polypeptide chain with a new combination of variable domains.
The nucleic acid may comprise, between nucleic acid encoding the first domain and nucleic acid encoding the second domain, nucleic acid encoding a site for recombination in vivo or in vitro.
For instance the loxP site, a 34 bp site at which recombination is catalysed by the protein Cre (Hoess et al., PNAS USA 79: 3398-3402, 1982, and Stemberg et al., J. Miol. Biol.; 150: 467-486, 1981).
This system has been used in the preparation of antibodies displayed on phage (P. Waterhouse et al., Nuc. Acid Research 21: 2265-2266, 1993; and WO93/19172).
The invention also provides a vector comprising such nucleic acid. The vector may comprise nucleic acid necessary for expression of the polypeptide. The vector may comprise nucleic acid for secretion of the polypeptide upon expression.
The vector may comprise nucleic acid encoding two of said polypeptides, a first polypeptide and a second polypeptide. There may be, between nucleic acid encoding the first polypeptide and nucleic acid encoding the second polypeptide, nucleic acid comprising a site for recombination in vivo or in vitro.
According to a futher aspect of the present invention there is provided a vector comprising nucleic acid encoding a first polypeptide and nucleic acid encoding a second polypeptide, each of the polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide but incapable of associating with it to form an antigen binding site, the nucleic acid encoding the first polypeptide being linked to nucleic acid encoding a signal sequence for export of the first polypeptide from a host cell upon expression, the nucleic acid encoding the second polypeptide being linked to nucleic acid encoding a signal sequence for export of the second polypeptide from a host cell upon expression and nucleic acid encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle.
Expression from such a vector will produce, on the surface of a bacteriophage particle a dimer of the two first and second polypeptides, one being attached to the particle by means of the surface component, the other being associated with it. This allows selection of displayed diabodies, with encoding nucleic acid, packaged within particles being easily isolated. Techniques of this kind are described in WO 92/01047.
Expression of a repertoire of diabodies and their display on secreted replicable genetic display packages, such as bacteriophage, is valuable for selection of binders for an antigen of interest from among many different possible combinations of heavy and light chainxe2x80x94many different possible binding specificities.
In another aspect the invention provides a vector comprising:
(a) nucleic acid encoding a first polypeptide domain, which comprises a binding region of an immunoglobulin heavy chain variable region, and a second polypeptide domain, which comprises a binding region of an immunoglobulin light chain variable region; and
(b) nucleic acid encoding a surface component of a filamentous bacteriophage;
expression of nucleic acid (a) producing a polypeptide (xe2x80x9cAxe2x80x9d) comprising the first and second domains linked but incapable of associating with each other to form an antigen binding site;
expression of nucleic acid (a) together with nucleic acid (b) producing a polypeptide (xe2x80x9cBxe2x80x9d) comprising the first and second domains linked but incapable of associating with each other to form an antigen binding site, fused to a surface component of a filamentous bacteriophage for display of the polypeptide (B) on the surface of bacteriophage particles;
nucleic acid (b) being linked to nucleic acid (a) by nucleic acid including an intervening suppressible stop codon.
A suppressible stop codon allows the translation of nucleotide sequences downstream of the codon in suppressor host cells but in non-suppressor cells translation may end at the codon. Examples of suppressible translational stop codons are the amber, ochre and opal codons. In fact, in most suppressor host cells, such as SupE cells, there is some xe2x80x9cslippagexe2x80x9d so there is some translation beyond the suppressible stop and some translation stopping at the stop.
This is useful, because it allow a vector according to this aspect of the present invention to be used to express both soluble polypeptides and polypeptides fused to a component of a bacteriophage, or other display package, for association of a dimer on the particle surface. Preferably the nucleic acid of the vector includes nucleic acid encoding secretory leader peptides or other signal sequences, so that the polypeptides are secreted from the cytosol of host cells into the periplasm (Skerra et al., Science 240: 1038: 1041, 1988; Better et al., Science 240: 1041-1043, 1988; WO92/01047).
The present invention also encompasses host cells transfected with any vector according to the present invention. Where the vector is one which has a suppressible stop codon as described, preferably the host cell is capable of providing conditions for co-expression of both polypeptides A and B (as discussed ie one which is VH-VL and one which is VH-VL fused to a surface component of a bacteriophage or other suitable organism).
The invention also provides a method of making polypeptides, dimers or other multimers according to the invention, which comprises culturing a host cell transfected with a vector under conditions for expression of the polypeptide(s), which may be conditions for co-expresssion of the said polypeptides A and B. The method may involve recovery of the polypeptide(s), dimer(s) etc from the host cell culture, either from the host cell or the medium. Recovery from the host cell may be from the periplasm, following secretion from the cytoplasm, or from inclusion bodies. Refolding from denaturing condition may be needed. The recovery is preferably by selection by binding with antigen of interest.
Many uses of the polypeptides according to the invention are envisaged, some of which are described infra. One use of particular interest is in assay for antigen, either homogeneous assay or heterogeneous assay.
In another aspect of the invention, a bispecific dimer is provided having (i) a first polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, and a polypeptide linker linking the first and second domains and allowing association of the domains with each other to form an antigen binding site; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, and a polypeptide linker linking the first and second domains and allowing association of the domains with each other to form an antigen binding site. The first domain of the first polypeptide and the second domain of the second polypeptide associate to form an antigen binding site which has a first binding specificity, while the second domain of the first polypeptide and the first domain of the second polypeptide associating to form an antigen binding site which has a second binding specificity.
In another aspect of the present invention there is provided a dimer of
(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the domains of the first polypeptide being linked but incapable of associating with each other to form an antigen binding site; and
(ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, and a polypeptide linker linking the first and second domains and allowing association of the domains of the second polypeptide with each other to form an antigen binding site;
the first domain of the first polypeptide and the second domain of the second polypeptide associating to form an antigen binding site; and
the second domain of the first polypeptide and the first domain of the second polypeptide associating to form an antigen binding site.
Such dimers may be bivalent or bispecific.
Various modifications to the polypeptides of the dimer may be made, in the same manner as is described supra for other aspects of the invention. Likewise, the present invention encompasses vectors comprising nucleic acid which encodes first and second polypeptides which are able to form a dimer according to this aspect of the invention, host cells transfected with such vectors and methods which comprise culturing host cells under conditions for expression of the first and second polypeptides, and recovery of a dimer which has bispecificity.
Polypeptides, multimers, nucleic acid, vectors, repertoires etc. disclosed by the experimental Examples infra are provided as aspects of the present invention.
instance the present invention also provides molecules which bind any of the following:a cell surface protein, a Fc receptor, a tumour specific marker, CEA, a virus HIV 1, a small chemical molecule, a hormone, a cytokine, TNFxcex1, an antibody, the idiotope of an antibody.
Dimers wherein the binding of an antigen to one antigen binding site of the dimer affects the binding of an antigem to the other binding site of the dimer are provided.
Those dimers wherein alterations of the linker lead to an improvement in antigen binding affintiy of at least one of the binding sites of the dimer are encompassed by the present invention.
Assays, which may be diagnostic, are provided, and may involve crosslinking of antigen or agglutination of cells. They may involve the binding of one antigen on a surface and the binding of an antigen in solution, and may involve detection using surface plasmon resonance. Molucules provided by embodiments of the present invention may bind to antigens on two different cell surfaces.
Additionally, the present invention provides pharmaceuticals comprising the polypeptides or multimers of the present invention, and the use of the polypeptides or multimers in the preparation of medicaments. Methods of treatment using polypeptides or multimers according to the present invention are encompassed.
Many other aspects of the present invention are discussed infra. Others will be apparent to those of ordinary skill in the art.
The present invention will now be discussed further by way of illustration and exemplification, with some comparison with existing art in the field of bivalent and bispecific antibodies and antibody fragments. What follows should not be interpreted as limiting the invention in any way. The following figures are mentioned (additional keys at end of the description):